Interleukin-1 β converting enzyme like apoptotic protease-6

ABSTRACT

Human ICE LAP-6 polypeptides and DNA (RNA) encoding such ICE LAP-6 and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such ICE LAP-6 for the treatment of a susceptibility to viral infection, tumorogenesis and to diseases and defects in the control embryogenesis and tissue homeostasis, and the nucleic acid sequences described above may be employed in an assay for ascertaining such susceptibility. Antagonists against such ICE LAP-6 and their use as a therapeutic to treat Alzheimer&#39;s disease, Parkinson&#39;s disease, rheumatoid arthritis, septic shock, sepsis, stroke, chronic inflammation, acute inflammation, CNS inflammation, osteoporosis, ischemia reperfusion injury, cell death associated with cardiovascular disease, polycystic kidney disease, apoptosis of endothelial cells in cardiovascular disease, degenerative liver disease, MS, ALS, cererbellar degeneration, ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes, aplastic anemia, male pattern baldness, and head injury damage are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding the interleukin-1 beta converting enzyme apoptosis proteases and for detecting altered levels of the polypeptide in a host.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.60/018,961 filed Jun. 5, 1996, 60/020,344 filed May 23, 1996 and60/017,949 filed May 20, 1996.

FIELD OF INVENTION

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of human interleukin-1 beta convertingenzyme apoptosis protease-6, hereinafter referred to as "ICE LAP-6".

BACKGROUND OF THE INVENTION

It has recently been discovered that an interleukin-1β converting enzyme(ICE) is responsible for cleaving pro-IL-1β into mature and active IL-1βand is also responsible for programmed cell death (or apoptosis), whichis a process through which organisms get rid of unwanted cells.

Apoptosis, or programmed cell death, is a physiologic process importantin the normal development and homeostasis of metazoans

In the nematode Caenorhabditis elegans, a genetic pathway of programmedcell death has been identified (Ellis, R. E., et al. Annu. Rev. CellBiol., 7:663-698 (1991)). Two genes, ced-3 and ced-4, are essential forcells to undergo programmed cell death in C. elegans (Ellis, H. M., andHorvitz, H. R., Cell, 44:817-829 (1986)). It is becoming apparent that aclass of cysteine proteases homologous to Caenorhabditis elegans Ced-3play the role of "executioner" in the apoptotic mechanism (Martin, S.J., and Green, D. R. (1995) Cell 82, 349-352; Chinnaiyan, A. a. D., VM.(1996) Current Biology 6; Henkart, P. (1996) Immunity 4, 195-201).Recessive mutations that eliminate the function of these two genesprevent normal programmed cell death during the development of C.elegans. The known vertebrate counterpart to ced-3 protein is ICE. Theoverall amino acid identity between ced-3 and ICE is 28%, with a regionof 115 amino acids (residues 246-360 of ced-3 and 164-278 of ICE) thatshows the highest identity (43%). This region contains a conservedpentapeptide, QACRG (SEQ ID NO:10) (residues 356-360 of ced-3), whichcontains a cysteine known to be essential for ICE function.

The similarity between ced-3 and ICE suggests not only that ced-3 mightfunction as a cysteine protease but also that ICE might act as avertebrate programmed cell death gene. ced-3 and the vertebratecounterpart, ICE, control programmed cell death during embryonicdevelopment, (Gagliamini, V. et al., Science, 263:826:828 (1994).

Mutations of ced-3 and ced-4 abolish the apoptotic capability of cellsthat normally die during C. elegans embryogenesis (Yuan, J. Y., andHorvitz, H. R. (1990) Dev Biol 138, 33-41). While no mammalian homologsof ced-4 have been identified, ced-3 shares sequence similarity withinterleukin-1b converting enzyme (ICE) (Yuan, J. et al(1993) Cell 75,641-652), a cysteine protease involved in the processing and activationof pro-IL-1b to an active cytokine (Cerretti, D. P., et al (1992)Science 256, 97-100, Thornberry, N. A.,et al (1992) Nature 356,768-774). Recently, numerous homologs of ICE/Ced-3 have beencharacterized, comprising a new gene family of cysteine proteases. Todate, seven members of the ICE/Ced-3 family have been identified andinclude ICE (Cerretti, D. P., et al (1992) Science 256, 97-100),TX/ICH2/ICE rel-II (Munday, N. A., et al (1995) J Biol Chem 270,15870-15876; Faucheu, C. et al. (1995) Embo J 14, 1914-1922; Kamens, Jet al.(1995) J Biol Chem 270, 15250-1525612), ICE rel-III (Munday, N.A., et al (1995) J Biol Chem 270, 15870-15876), ICH1/Nedd-2 (Kumar, S.,et al. (1994) Genes and Development 8, 1613-1626; Wang, L., et al.(1994) Cell 78, 739-750), Yama/CPP32/Apopain (Tewari, M., et al. (1995)Cell 81, 801-809; Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem.269, 30761-30764; Nicholson, D. Wet al. (1995) Nature 376, 37-43), Mch2Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 30761-30764)and ICE-LAP3/Mch3/CMH-1 (Duan, H., et al. (1996) J. Biol. Chem. 271,35013-35035; Fernandes-Alnemri, T., et al. (1995) Cancer Research 55,6045-6052; Lippke, J. A., et al. (1996) The Journal of BiologicalChemistry 271, 1825-1828). All family members share sequence homologywith ICE/Ced-3 and contain an active site QACRG (SEQ ID NO:10)pentapeptide in which the cysteine residue is catalytic. Ectopicexpression of these proteases in a variety of cells causes apoptosis.Phylogenetic analysis of the ICE/ced-3 gene family revealed threesubfamilies (Chinnaiyan, A. a. D., VM. (1996) Current Biology 6; uan,H., et al. (1996) J. Biol. Chem. 271, 35013-35035). Yama, ICE-LAP3, andMch2 are closely related to C.elegans Ced-3 and comprise the Ced-3subfamily. ICE and the ICE-related genes, ICE rel II, and ICE rel IIIform the ICE subfamily, while ICH1 and its mouse homologue, NEDD-2 formthe NEDD-2 subfamily. Based on similarities with the structuralprototype interleukin-1b converting enzyme, ICE/Ced-3 family members aresynthesized as zymogens that are capable of being processed to formactive heterodimeric enzymes (Thomberry, N. A.,et al (1992) Nature 356,768-774). It will be important to determine which family members are infact activated in response to apoptotic stimuli. Previous studies havedemonstrated that pro-Yama and pro-ICE-LAP3 are processed into activesubunits in response to various death stimuli including engagement ofFas/APO-1 or treatment with staurosporine (Duan, H., et al. (1996) J.Biol. Chem. 271, 35013-35035; Chinnaiyan, A. M., et al. 1996) Journal ofBiological Chemistry 271, 4573-4576). Further, the serine proteasegranzyme B, one of the major effectors of cytotoxic T cell-mediatedapoptosis, was shown to directly activate Yama (but not ICE), in vitro(Quan, L. T., et al. (1996) PNAS 93, In Press; Darmon, A. J., et al.(1995) Nature 377, 446-448).

ICE mRNA has been detected in a variety of tissues, including peripheralblood monocytes, peripheral blood lymphocytes, peripheral bloodneutrophils, resting and activated peripheral blood T lymphocytes,placenta, the B lymphoblastoid line CB23, and monocytic leukemia cellline THP-1 cells (Cerretti, D. P., et al., Science, 256:97-100 (1992)),indicating that ICE may have an additional substrate in addition topro-IL-1. The substrate that ICE acts upon to cause cell death ispresently unknown. One possibility is that it may be a vertebratehomolog of the C. elegans cell death gene ced-4. Alternatively, ICEmight directly cause cell death by proteolytically cleaving proteinsthat are essential for cell viability.

The mammalian gene bcl-2, has been found to protect immune cells calledlymphocytes from cell suicide. Also, crmA, a cow pox virus gene proteinproduct inhibits ICE's protein splitting activity.

Clearly, there is a need for factors that are useful for inducingapoptosis for therapeutic purposes, for example, as an antiviral agent,an anti-tumor agent and to control embryonic development and tissuehomeostasis, and the roles of such factors in dysfunction and disease.Further, there is clear a need for factors that are useful for reducingor halting apoptosis for therapeutic purposes, for example, to treatdiseases caused or associated with apoptosis, such as, particularlyAlzheimer's disease, Parkinson's disease, rheumatoid arthritis, septicshock, sepsis, stroke, chronic inflammation, acute inflammation, CNSinflammation, osteoporosis, ischemia reperfusion injury, cell deathassociated with cardiovascular disease, polycystic kidney disease,apoptosis of endothelial cells in cardiovascular disease, degenerativeliver disease, MS, ALS, cererbellar degeneration, ischemic injury,myocardial infarction, AIDS, myelodysplastic syndromes, brain damage,aplastic anemia, male pattern baldness, and head injury damage. There isa need, therefore, for identification and characterization of suchfactors that are interleukin-1 beta converting enzyme apoptosisproteases, and which can play a role in preventing, ameliorating orcorrecting dysfunctions or diseases.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelICE LAP-6 by homology between the amino acid sequence set out in FIG. 1or the polypeptide encoded by the deposited clone and known amino acidsequences of other proteins of the mammalian ICE/ced-3 family and the C.elegans Ced-3 protein.

It is a further object of the invention, moreover, to providepolynucleotides that encode ICE LAP-6, particularly polynucleotides thatencode the polypeptide herein designated ICE LAP-6 and thepolynucleotide of the deposited clone.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding human ICE LAP-6 setforth in FIGS. 2A, 2B, and 2C.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA in FIGS.2A, 2B, and 2C or derived using theprimers set forth in Example 1, or a polynucleotide encoding thepolypeptide in FIG. 1 or derived from the polypeptide encoded by thedeposited clone.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding human ICE LAP-6, includingmRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect ofthe invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human ICE LAP-6.

It also is an object of the invention to provide ICE LAP-6 polypeptides,particularly human ICE LAP-6 polypeptides, that may be employed fortherapeutic purposes, for example, to treat viral infection, as ananti-tumor agent and to control embryonic development and tissuehomeostasis.

In accordance with this aspect of the invention there are provided novelpolypeptides of human origin referred to herein as ICE LAP-6 as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human ICE LAP-6 encoded by naturally occurringalleles of the human ICE LAP-6 gene.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned ICE LAP-6 polypeptidescomprising culturing host cells having expressibly incorporated thereinan exogenously-derived human ICE LAP-6-encoding polynucleotide underconditions for expression of human ICE LAP-6 in the host and thenrecovering the expressed polypeptide. ICE LAP-6 may also be purifiedfrom natural sources using any of many well known techniques.

In accordance with another object the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical, diagnostic and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing ICE LAP-6 expression in cellsby determining ICE LAP-6 polypeptides or ICE LAP-6-encoding mRNA; as anantiviral agent, an anti-tumor agent and to control embryonicdevelopment and tissue homeostasis in vitro, ex vivo or in vivo byexposing cells to ICE LAP-6 polypeptides or polynucleotides as disclosedherein; assaying genetic variation and aberrations, such as defects, inICE LAP-6 genes; and administering an ICE LAP-6 polypeptide orpolynucleotide to an organism to augment ICE LAP-6 function or remediateICE LAP-6 dysfunction. Agonists targeted to defective cellularproliferation, including, for example, cancer cell and solid tumor cellproliferation, may be used for the treatment of these diseases. Suchtargeting may be achieved via gene therapy using antibody fusions.Agonists may also be used to treat follicular lymphomas, carcinomasassociated with p53 mutations, autoimmune disorders, such as, forexample, SLE, immune-mediated glomerulonephritis; and hormone-dependenttumors, such as, for example, breast cancer, prostate cancer and ovarycancer; and viral infections, such as, for example, herpesviruses,poxviruses and adenoviruses.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided probes that hybridize tohuman ICE LAP-6 sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against ICE LAP-6 polypeptides.In certain particularly preferred embodiments in this regard, theantibodies are highly selective for human ICE LAP-6.

In accordance with another aspect of the present invention, there areprovided ICE LAP-6 agonists. Among preferred agonists are molecules thatmimic ICE LAP-6, that bind to ICE LAP-6-binding molecules or receptormolecules, and that elicit or augment ICE LAP-6-induced responses. Alsoamong preferred agonists are molecules that interact with ICE LAP-6 orICE LAP-6 polypeptides, or with other modulators of ICE LAP-6 activitiesand/or gene expression, and thereby potentiate or augment an effect ofICE LAP-6 or more than one effect of ICE LAP-6.

In accordance with yet another aspect of the present invention, thereare provided ICE LAP-6 antagonists. Among preferred antagonists arethose which mimic ICE LAP-6 so as to bind to ICE LAP-6 receptor orbinding molecules but not elicit an ICE LAP-6-induced response or morethan one ICE LAP-6-induced response. Also among preferred antagonistsare molecules that bind to or interact with ICE LAP-6 so as to inhibitan effect of ICE LAP-6 or more than one effect of ICE LAP-6 or whichprevent expression of ICE LAP-6.

In a further aspect of the invention there are provided compositionscomprising an ICE LAP-6 polynucleotide or an ICE LAP-6 polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprisean ICE LAP-6 polynucleotide for expression of an ICE LAP-6 polypeptidein a host organism for treatment of disease. Particularly preferred inthis regard is expression in a human patient for treatment of adysfunction associated with aberrant endogenous activity of ICE LAP-6.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 [SEQUENCE ID NO. 1] shows the predicted amino acid sequence ofhuman ICE LAP-6. The active site pentapeptide QACGG (SEQ ID NO:11) isunderlined. Putative amino acid (Asp) cleavage sites are indicated withbold letters.

FIGS. 2A, 2B, and 2C [SEQUENCE ID NO. 2] show a nucleic acid sequence ofhuman ICE LAP-6.

FIG. 3 [SEQUENCE ID NO. 3] shows a nucleic acid sequence variant derivedfrom human ICE LAP-6.

FIG. 4 [SEQUENCE ID NO. 4] shows an amino acid sequence variant derivedfrom human ICE LAP-6.

FIG. 5 shows phylogenetic analysis of the ICE/ced-3 gene family.

FIG. 6 shows MCF7 the results of an analysis of breast carcinoma cellstransiently transfected demonstrating that over-expression of ICE LAP-6induces cell death in mammalian cells.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notlimitative of the invention.

DIGESTION of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes referred to herein are commerciallyavailable and their reaction conditions, cofactors and otherrequirements for use are known and routine to the skilled artisan.

For analytical purposes, typically, 1 μg of plasmid or DNA fragment isdigested with about 2 units of enzyme in about 20 μl of reaction buffer.For the purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzymein proportionately larger volumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates transcription ortranslation or other processes important to expression of thepolypeptide in a host cell, or a polynucleotide comprising both a regionthat encodes a polypeptide and a region operably linked thereto thatregulates expression.

Genetic elements may be comprised within a vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome; not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo polypeptides as determined by comparing the amino acid sequence andits conserved amino acid substitutes of one polypeptide to the sequenceof a second polypeptide. Moreover, also known in the art is "identity"which means the degree of sequence relatedness between two polypptide ortwo polynucleotides sequences as determined by the identity of the matchbetween two strings of such sequences. Both identity and similarity canbe readily calculated (Computational Molecular Biology, Lesk, A.M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). While there exist a number of methods to measureidentity and similarity between two polynucleotide or polypeptidesequences, the terms "identity" and "similarity" are well known toskilled artisans (Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonlyemployed to determine identity or similarity between two sequencesinclude, but are not limited to disclosed in Carillo, H., and Lipman,D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods todetermine identity are designed to give the largest match between thetwo sequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not"isolated," but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is "isolated", as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

LIGATION refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) ("Sambrook") and Maniatis et al., pg. 146, as citedbelow.

OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often theterm refers to single-stranded deoxyribonucleotides, but it can refer aswell to single-or double-stranded ribonucleotides, RNA:DNA hybrids anddouble-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5' phosphate. The 5' ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3' end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, readily will form a phosphodiester bond with a 5' phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5' phosphates of the other polynucleotide(s) prior toligation.

PLASMIDS generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart.

Starting plasmids disclosed herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids by routine application of well known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single-and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

In addition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are"polynucleotides" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Analysis for protein modifications andnonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcell often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

(1) A polynucleotide that differs in nucleotide sequence from another,reference polynucleotide. Generally, differences are limited so that thenucleotide sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

(2) A polypeptide that differs in amino acid sequence from another,reference polypeptide. Generally, differences are limited so that thesequences of the reference and the variant are closely similar overalland, in many region, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

RECEPTOR MOLECULE, as used herein, refers to molecules which bind orinteract specifically with ICE LAP-6 polypeptides of the presentinvention, including not only classic receptors, which are preferred,but also other molecules that specifically bind to or interact withpolypeptides of the invention (which also may be referred to as "bindingmolecules" and "interaction molecules," respectively and as "ICE LAP-6binding molecules" and "ICE LAP-6 interaction molecules." Bindingbetween polypeptides of the invention and such molecules, includingreceptor or binding or interaction molecules may be exclusive topolypeptides of the invention, which is very highly preferred, or it maybe highly specific for polypeptides of the invention, which is highlypreferred, or it may be highly specific to a group of proteins thatincludes polypeptides of the invention, which is preferred, or it may bespecific to several groups of proteins at least one of which includespolypeptides of the invention.

Receptors also may be non-naturally occurring, such as antibodies andantibody-derived reagents that bind specifically to polypeptides of theinvention.

Description of the Invention

The present invention relates to novel ICE LAP-6 polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human ICE LAP-6, which is related by aminoacid sequence homology to human interleukin-1 beta converting enzymeapoptosis protease polypeptides. The invention relates especially to ICELAP-6 having the nucleotide and amino acid sequences set out in FIGS. 1and 2A, 2B and 2C respectively. It will be appreciated that thenucleotide and amino acid sequences set out in FIGS. 2A, 2B and 2Crespectively, were obtained by sequencing the cDNA obtained from a humanK562 (erythroleukemia) cell line cDNA library.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the ICE LAP-6 polypeptidehaving the deduced amino acid sequence of FIG. 1 or the polypeptideencoded by the deposited clone.

In accordance with another aspect of the present invention, there areprovided isolated polynucleotides which encode the ICE LAP-6 polypeptidehaving the deduced amino acid sequence of FIG. 1 or the polypeptideencoded by the deposited clone.

Using the information provided herein, such as the polynucleotide primersequences set out in Example 1, a polynucleotide of the presentinvention encoding human ICE LAP-6 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA from cells from human neutrophils and kidney tissue asstarting material. Illustrative of the invention, the polynucleotide ofthe invention was discovered as described in Example 1. ICE LAP 7 canalso be obtained from other tissues and cDNA libraries, for example,libraries derived from cells of human cells, tissue and cell lines suchas, activated human neutrophil, erythroleukemia and kidney cells.

Human ICE LAP-6 of the invention is structurally related to otherproteins of the human interleukin-1 beta converting enzyme apoptosisprotease family, as shown by the results of sequencing the cDNA encodinghuman ICE LAP-6 in FIG. 1. The cDNA of FIGS. 2A, 2B and 2C was obtainedas described in Example 1. The polypeptide of FIG. 1 and the polypeptideencoded by the deposited clone each are proteins which have a deducedmolecular weight of about 45.8 kDa.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide derived using the primers setforth in Example 1, or the polynucleotide of FIGS. 2A, 2B, and 2C. Italso may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of the cDNA of FIG. 1 or the polypeptide encoded by thedeposited clone.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 1 or the polypeptide encoded by the deposited clone may include,but are not limited to the coding sequence for the mature polypeptide,by itself; the coding sequence for the mature polypeptide and additionalcoding sequences, such as those encoding a leader or secretory sequence,such as a pre-, or pro- or prepro- protein sequence; the coding sequenceof the mature polypeptide, with or without the aforementioned additionalcoding sequences, together with additional, non-coding sequences,including for example, but not limited to introns and non-coding 5' and3' sequences, such as the transcribed, non-translated sequences thatplay a role in transcription, mRNA processing--including splicing andpolyadenylation signals, for example--ribosome binding and stability ofmRNA; additional coding sequence which codes for additional amino acids,such as those which provide additional functionalities. Thus, forinstance, the polypeptide may be fused to a marker sequence, such as apeptide, which facilitates purification of the fused polypeptide. Incertain preferred embodiments of this aspect of the invention, themarker sequence is a hexa-histidine peptide, such as the tag provided inthe pQE vector (Qiagen, Inc.), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. The HA tag corresponds toan epitope derived of influenza hemagglutinin protein, which has beendescribed by Wilson et al., Cell 37: 767 (1984), for instance.

Also provided is mature ICE LAP-6 processed from its precursor molecule,via autocatalysis or by other enzymes, to produce two subunits, whichform an active heterodimer (both subunits) or tetramer (two sets of suchheterodimers).

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlythe human ICE LAP-6 having the amino acid sequence set out in FIG. 1 orthe amino acid sequence of the polypeptide encoded by the depositedclone. The term encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by introns) together with additional regions, thatalso may contain coding and/or non-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or the amino acid sequence of the polypeptide encoded by thedeposited clone. A variant of the polynucleotide may be a naturallyoccurring variant such as a naturally occurring allelic variant, or itmay be a variant that is not known to occur naturally. Suchnon-naturally occurring variants of the polynucleotide may be made bymutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of ICE LAP-6 set out in FIG. 1 or the amino acid sequence ofthe polypeptide encoded by the deposited clone; variants, analogs,derivatives and fragments thereof, and fragments of the variants,analogs and derivatives.

Further particularly preferred in this regard are polynucleotidesencoding ICE LAP-6 variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the ICE LAP-6 polypeptide of FIG. 1 or the polypeptideencoded by the deposited clone in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these aresubstitutions, additions and deletions, which do not alter theproperties and activities of the ICE LAP-6. Also especially preferred inthis regard are conservative substitutions. Most highly preferred arepolynucleotides encoding polypeptides having the amino acid sequence ofFIG. 1 or the amino acid sequence of the polypeptide encoded by thedeposited clone, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical to a polynucleotide encoding the ICE LAP-6polypeptide having the amino acid sequence set out in FIG. 1 or theamino acid sequence of the polypeptide encoded by the deposited clone,and polynucleotides which are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical to a polynucleotide encoding theICE LAP-6 polypeptide of the human cDNA and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and among theseparticularly preferred polynucleotides, those with at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNA of

FIGS. 2A, 2B, and 2C encoded by the polynucleotide sequence of thedeposited clone, or derived using the primers set forth in Example 1.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for cDNA and genornic DNA toisolate full-length cDNAs and genomic clones encoding ICE LAP-6 and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the human ICE LAP-6 gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

For example, the coding region of the ICE LAP-6 gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofhuman cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays, inter alia.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited Materials

A deposit containing a human ICE LAP-6 cDNA has been deposited with theAmerican Type Culture Collection, as noted above. Also as noted above,the human cDNA deposit is referred to herein as "the deposited clone" oras "the cDNA of the deposited clone."

The deposited clone was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 USA, onMay 30, 1996.

The deposited material is a pBluescript SK (-) plasmid (Stratagene, LaJolla, Calif.) that contains the full length ICE LAP-6 cDNA, referred toas "1095150" upon deposit.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a human ICE LAP-6 polypeptidewhich has the deduced amino acid sequence of FIG. 1 and the amino acidsequence of the the polypeptide encoded by the deposited clone.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms "fragment," "derivative" and "analog" whenreferring to the polypeptide of FIG. 1 or the polypeptide encoded by thedeposited clone, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or thepolypeptide encoded by the deposited clone each may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of ICE LAP-6 setout in FIG. 1 or the amino acid sequence of the polypeptide encoded bythe deposited clone, variants, analogs, derivatives and fragmentsthereof, and variants, analogs and derivatives of the fragments.Alternatively, particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of the ICELAP-6, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Lle; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the ICE LAP-6 polypeptideof FIG. 1 or the amino acid sequence of the polypeptide encoded by thedeposited clone, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are substitutions,additions and deletions, which do not alter the properties andactivities of the ICE LAP-6. Also especially preferred in this regardare conservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 1 or the amino acid sequence ofthe polypeptide encoded by the deposited clone without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 1 or the polypeptide encoded by the deposited clone (in particularthe mature polypeptide) as well as polypeptides which have at least 80%identity to the polypeptide of FIG. 1 or the polypeptide encoded by thedeposited clone and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 1 or thepolypeptide encoded by the deposited clone and still more preferably atleast 95% similarity (still more preferably at least 95% identity) tothe polypeptide of FIG. 1 or the polypeptide encoded by the depositedclone and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of ICE LAP-6, most particularlyfragments of the ICE LAP-6 having the amino acid set out in FIG. 1 orthe amino acid sequence of the polypeptide encoded by the depositedclone, and fragments of variants and derivatives of the ICE LAP-6 ofFIG. 1 or the polypeptide encoded by the deposited clone, such as, forexample the amino acid sequence of FIG. 4.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned ICE LAP-6 polypeptides and variants or derivativesthereof.

Such fragments may be "free-standing," i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of an ICE LAP-6polypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the ICE LAP-6fragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from ICE LAP-6.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5-15, 10-20, 15-40,30-55, 41-65, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113amino acids long.

In this context about includes the particularly recited range and rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes. For instance, about 40-90 aminoacids in this context means a polypeptide fragment of 40 plus or minusseveral, a few, 5, 4, 3, 2 or 1 amino acids to 90 plus or minus severala few, 5, 4, 3, 2 or 1 amino acid residues, i.e., ranges as broad as 40minus several amino acids to 90 plus several amino acids to as narrow as40 plus several amino acids to 90 minus several amino acids.

Highly preferred in this regard are the recited ranges plus or minus asmany as 5 amino acids at either or at both extremes. Particularly highlypreferred are the recited ranges plus or minus as many as 3 amino acidsat either or at both the recited extremes. Especially particularlyhighly preferred are ranges plus or minus 1 amino acid at either or atboth extremes or the recited ranges with no additions or deletions. Mosthighly preferred of all in this regard are fragments from about 5-15,10-20, 15-40, 30-55, 41-65, 41-80, 41-90, 50-100, 75-100, 90-115,100-125, and 110-113 amino acids long.

Among especially preferred fragments of the invention are truncationmutants of ICE LAP-6. Truncation mutants include ICE LAP-6 polypeptideshaving the amino acid sequence of FIG. 1 or the amino acid sequence ofthe polypeptide encoded by the deposited clone, or of variants orderivatives thereof, except for deletion of a continuous series ofresidues (that is, a continuous region, part or portion) that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus or, as in double truncation mutants, deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. Fragments having the size ranges setout about also are preferred embodiments of truncation fragments, whichare especially preferred among fragments generally.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of ICE LAP-6.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions("alpha-regions"), beta-sheet and beta-sheet-forming regions("beta-regions"), turn and turn-forming regions ("turn-regions"), coiland coil-forming regions ("coil-regions"), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of ICE LAP-6.

Among highly preferred fragments in this regard are those that compriseregions of ICE LAP-6 that combine several structural features, such asseveral of the features set out above. In this regard, the regionsdefined by the residues about 10 to about 20, about 40 to about 50,about 70 to about 90 and about 100 to about 113 of FIG. 1 or thepolypeptide encoded by the deposited clone, which all are characterizedby amino acid compositions highly characteristic of turn-regions,hydrophilic regions, flexible-regions, surface-forming regions, and highantigenic index-regions, are especially highly preferred regions. Suchregions may be comprised within a larger polypeptide or may be bythemselves a preferred fragment of the present invention, as discussedabove. It will be appreciated that the term "about" as used in thisparagraph has the meaning set out above regarding fragments in general.

Further preferred regions are those that mediate activities of ICELAP-6. Most highly preferred in this regard are fragments that have achemical, biological or other activity of ICE LAP-6, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Highly preferred in this regard are fragments thatcontain regions that are homologs in sequence, or in position, or inboth sequence and to active regions of related polypeptides, whichinclude human interleukin-1 beta converting enzyme apoptosis proteases.Among particularly preferred fragments in these regards are truncationmutants, as discussed above.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespondent to the preferred fragments, as discussed above. Preferredpolynucleotides fragments may be derived from the sequences of FIGS. 2A,2B, 2C and 3.

Vectors, Host Cells, Expression

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate, polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation also may be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. cited above, which is illustrative of the manylaboratory manuals that detail these techniques.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case viralpropagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such assimian virus 40 ("SV40"), vaccinia viruses, adenoviruses, fowl poxviruses, pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids, all maybe used for expression in accordance with this aspect of the presentinvention. Generally, any vector suitable to maintain, propagate orexpress polynucleotides to express a polypeptide in a host may be usedfor expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al. cited elsewhereherein.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous promoters not mentioned are suitable for use inthis aspect of the invention are well known and readily may be employedby those of skill in the manner illustrated by the discussion and theexamples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as repressor binding sites and enhancers, amongothers.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts for a greatvariety of expression constructs are well known, and those of skill willbe enabled by the present disclosure readily to select a host forexpressing a polypeptides in accordance with this aspect of the presentinvention.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9 , available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. These vectors are listed solelyby way of illustration of the many commercially available and well knownvectors that are available to those of skill in the art for use inaccordance with this aspect of the present invention. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of a polynucleotideor polypeptide of the invention in a host may be used in this aspect ofthe invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two such vectors are pKK232-8 and pCM7. Thus, promoters forexpression of polynucleotides of the present invention include not onlywell known and readily available promoters, but also promoters thatreadily may be obtained by the foregoing technique, using a reportergene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are thecytomegalovirus ("CMV") immediate early promoter, the HSV thymidinekinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous sarcoma virus ("RoSV"), andmetallothionein promoters, such as the mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as SAMBROOK.

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., cited above.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase("PGK"), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trp 1 gene of S. cerevisiae.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiating AUG. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal and a transcription termination signalappropriately disposed at the 3' end of the transcribed region.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, a region may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art.

Suitable prokaryotic hosts for propagation, maintenance or expression ofpolynucleotides and polypeptides in accordance with the inventioninclude Escherichia coli, Bacillus subtilis and Salmonella typhimurium.Various species of Pseudomonas, Streptomyces, and Staphylococcus aresuitable hosts in this regard. Moreover, many other hosts also known tothose of skill may be employed in this regard.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wisc.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the COS-6lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23:175 (1981). Other cell lines capable of expressing a compatible vectorinclude for example, the C 127, 3T3, CHO, HeLa, human kidney 293 and BHKcell lines.

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments in this regard DNA sequences derived from the SV40 splicesites, and the SV40 polyadenylation sites are used for requirednon-transcribed genetic elements of these types.

The ICE LAP-6 polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography ("HPLC") is employed for purification. Well knowntechniques for refolding protein may be employed to regenerate activeconformation when the polypeptide is denatured during isolation and orpurification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

ICE LAP-6 polynucleotides and polypeptides may be used in accordancewith the present invention for a variety of applications, particularlythose that make use of the chemical and biological properties of ICELAP-6. Additional applications relate to diagnosis and to treatment ofdisorders of cells, tissues and organisms. These aspects of theinvention are illustrated further by the following discussion.

Polynucleotide Assays

This invention is also related to the use of the ICE LAP-6polynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form of ICELAP-6 associated with a dysfunction will provide a diagnostic tool thatcan add or define a diagnosis of a disease or susceptibility to adisease which results from under-expression over-expression or alteredexpression of ICE LAP-6. Individuals carrying mutations in the human ICELAP-6 gene may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR prior to analysis. PCR (Saiki et al., Nature,324:163-166 (1986)). RNA or cDNA may also be used in the same ways. Asan example, PCR primers complementary to the nucleic acid encoding ICELAP-6 can be used to identify and analyze ICE LAP-6 expression andmutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled ICE LAP-6 RNA or alternatively, radiolabeled ICE LAP-6antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms ("RFLP") and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

In accordance with a further aspect of the invention, there is provideda process for determining disease associated with viral infection,tumorogenesis and to control embryogenesis and tissue homeostasis.Diseases associated with viral infection, tumorogenesis and to controlembryogenesis and tissue homeostasis, or a susceptibility to viralinfection, tumorogenesis and to diseases and defects in the control ofcontrol of embryogenesis and tissue homeostasis. Thus, a mutation in ICELAP-6 indicates a susceptibility to viral infection, tumorogenesis andto diseases and defects in the control embryogenesis and tissuehomeostasis, and the nucleic acid sequences described above may beemployed in an assay for ascertaining such susceptibility. Thus, forexample, the assay may be employed to determine a mutation in a humanICE LAP-6 protein as herein described, such as a deletion, truncation,insertion, frame shift, etc., with such mutation being indicative of asusceptibility to viral infection, tumorogenesis and to diseases anddefects in the control of embryogenesis and tissue homeostasis.

A mutation may be ascertained for example, by a DNA sequencing assay.Tissue samples, including but not limited to blood samples are obtainedfrom a human patient. The samples are processed by methods known in theart to capture the RNA. First strand cDNA is synthesized from the RNAsamples by adding an oligonucleotide primer consisting of polythymidineresidues which hybridize to the polyadenosine stretch present on themRNA's. Reverse transcriptase and deoxynucleotides are added to allowsynthesis of the first strand cDNA. Primer sequences are synthesizedbased on the DNA sequence of the ICE LAP-6 protein of the invention. Theprimer sequence is generally comprised of at least 15 consecutive bases,and may contain at least 30 or even 50 consecutive bases.

Individuals carrying mutations in the gene of the present invention mayalso be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, includingbut not limited to blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166(1986)) prior to analysis. RT-PCR can also be used to detect mutations.It is particularly preferred to used RT-PCR in conjunction withautomated detection systems, such as, for example, GeneScan. RNA or cDNAmay also be used for the same purpose, PCR or RT-PCR. As an example, PCRprimers complementary to the nucleic acid encoding ICE LAP-6 can be usedto identify and analyze mutations. For example, deletions and insertionscan be detected by a change in size of the amplified product incomparison to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to radiolabeled RNA or alternatively,radiolabeled antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Primers, selected by well known methods, may be used for amplifying ICELAP-6 cDNA isolated from a sample derived from a patient. The inventionalso provides the primers selected with 1, 2, 3 or 4 nucleotides removedfrom the 5' and/or the 3' end. The primers may be used to amplify thegene isolated from the patient such that the gene may then be subject tovarious techniques for elucidation of the DNA sequence. In this way,mutations in the DNA sequence may be diagnosed.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence and/or quantitation ofthe level of the sequence may be achieved by methods such ashybridization, RNase protection, chemical cleavage, direct DNAsequencing or the use of restriction enzymes, (e.g., RestrictionFragment Length Polymorphisms (RFLP)) and Southern blotting of genomicDNA.

The invention provides a process for diagnosing or detecting, disease,particularly Alzheimer's disease, Parkinson's disease, rheumatoidarthritis, septic shock, sepsis, stroke, chronic inflammation, acuteinflammation, CNS inflammation, osteoporosis, ischemia reperfusioninjury, cell death associated with cardiovascular disease, polycystickidney disease, apoptosis of endothelial cells in cardiovasculardisease, degenerative liver disease, MS, ALS, cererbellar degeneration,ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes,aplastic anemia, male pattern baldness, and head injury damage, as wellas a susceptibility to viral infection and cancer, an to detect aberrantcontrol of embryonic development and tissue homeostasis, comprisingdetermining from a sample derived from a patient altered expression ofpolynucleotide having the sequence of FIG. 1 or the polynucleotidesequence of the deposited clone as compared to normal control samples.Expression of polynucleotide can be measured using any one of themethods well known in the art for the quantation of polynucleotides,such as, for example, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomallocation.

As an example of how this is performed, ICE LAP-6 DNA is digested andpurified with QIAEX II DNA purification kit (QIAGEN, Inc., Chatsworth,Calif.) and ligated to Super Cos 1 cosmid vector (STRATAGENE, La Jolla,Calif.). DNA is purified using Qiagen Plasmid Purification Kit (QIAGENInc., Chatsworth, Calif.) and 1 mg is labeled by nick translation in thepresence of Biotin-dATP using BioNick Labeling Kit (GibcoBRL, LifeTechnologies Inc., Gaithersburg, Md.). Biotinilation is detected withGENE-TECT Detection System (CLONTECH Laboratories, Inc. Palo Alto,Calif.). In situ Hybridization is performed on slides using ONCOR LightHybridization Kit (ONCOR, Gaithersberg, Md.) to detect single copysequences on metaphase chromosomes. Peripheral blood of normal donors iscultured for three days in RPMI 1640 supplemented with 20% FCS, 3% PHAand penicillin/streptomycin, synchronized with 10⁻⁶ M methotrexate for17 hours and washed twice with unsupplemented RPMI. Cells are incubatedwith 10⁻³ M thymidine for 7 hours. The cells are arrested in metaphaseafter 20 minutes incubation with colcemid (0.5 μg/ml) followed byhypotonic lysis in 75 mM KCI for 15 minutes at 37° C. Cell pellets arethen spun out and fixed in Carnoy's fixative (3:1 methanouacetic acid).

Metaphase spreads are prepared by adding a drop of the suspension ontoslides and aid dried. Hybridization is performed by adding 100 ng ofprobe suspended in 10 ml of hybridization mix (50% formamide, 2×SSC, 1%dextran sulfate) with blocking human placental DNA 1 μg/ml), Probemixture is denatured for 10 minutes in 70° C. water bath and incubatedfor 1 hour at 37° C., before placing on a prewarmed (37° C.) slide,which is previously denatured in 70% formamide/2×SSC at 70° C., anddehydrated in ethanol series, chilled to 4° C.

Slides are incubated for 16 hours at 37° C. in a humidified chamber.Slides are washed in 50% formamide/2×SSC for 10 minutes at 41° C. and2xSSC for 7 minutes at 37° C. Hybridization probe is detected byincubation of the slides with FITC-Avidin (ONCOR, Gaithersberg, Md.),according to the manufacturer protocol. Chromosomes are counterstainedwith propridium iodine suspended in mounting medium. Slides arevisualized using a Leitz ORTHOPLAN 2-epifluorescence microscope and fivecomputer images are taken using Imagenetics Computer and Macintoshprinter.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (publicly available on-line via computer(Internet)). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis using well known methods.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of an ICE LAP-6 gene. This can beaccomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA is used forin situ chromosome mapping using well known techniques for this purpose.Typically, in accordance with routine procedures for chromosome mapping,some trial and error may be necessary to identify a genomic probe thatgives a good in situ hybridization signal.

In some cases, in addition, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3' untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization ("FISH") of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see Verma et al.,HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, NewYork (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,MENDELIAN INHERITANCE IN MAN (publicly available on line via computer).The relationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Polypeptide Assays

The present invention also relates to a quantitative andsemi-quantitative diagnostic assays for detecting levels of ICE LAP-6protein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of ICE LAP-6 proteincompared to normal control tissue samples may be used to detect thepresence of a tumor, or other abnormal cell growth or proliferation, forexample. Assay techniques that can be used to determine levels of aprotein, such as an ICE LAP-6 protein of the present invention, in asample derived from a host are well-known to those of skill in the art.Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis and ELISA assays. Among these ELISAsfrequently are preferred. An ELISA assay initially comprises preparingan antibody specific to ICE LAP-6, preferably a monoclonal antibody. Inaddition a reporter antibody generally is prepared which binds to themonoclonal antibody. The reporter antibody is attached a detectablereagent such as radioactive, fluorescent or enzymatic reagent, in thisexample horseradish peroxidase enzyme.

To carry out an ELISA a sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attach to any ICE LAP-6 proteins attached tothe polystyrene dish. Unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is placedin the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to ICE LAP-6. Unattached reporter antibody isthen washed out. Reagents for peroxidase activity, including acolorimetric substrate are then added to the dish. Immobilizedperoxidase, linked to ICE LAP-6 through the primary and secondaryantibodies, produces a colored reaction product. The amount of colordeveloped in a given time period indicates the amount of ICE LAP-6protein present in the sample. Quantitative results typically areobtained by reference to a standard curve.

A competition assay may be employed wherein antibodies specific to ICELAP-6 attached to a solid support and labeled ICE LAP-6 and a samplederived from the host are passed over the solid support and the amountof label detected attached to the solid support can be correlated to aquantity of ICE LAP-6 in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against ICE LAP-6 may be employed toinhibit the action of such ICE LAP-6 polypeptides, for example, in thetreatment of Alzheimer's disease, Parkinson's disease, rheumatoidarthritis, septic shock, sepsis, stroke, chronic inflammation, acuteinflammation, CNS inflammation, osteoporosis, ischemnia reperfusioninjury, cell death associated with cardiovascular disease, polycystickidney disease, apoptosis of endothelial cells in cardiovasculardisease, degenerative liver disease, MS, ALS, cererbellar degeneration,ischemic injury, myocardial infarction, AIDS, myelodysplastic syndromes,aplastic anemia, male pattern baldness, and head injury damage.

ICE LAP-6 Binding Molecules and Assays

This invention also provides a method for identification of molecules,such as receptor molecules, that bind ICE LAP-6. Genes encoding proteinsthat bind ICE LAP-6, such as receptor proteins, can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting. Such methods are described in many laboratorymanuals such as, for instance, Coligan et al., Current Protocols inImmunology 1(2): Chapter 5 (1991).

For instance, expression cloning may be employed for this purpose. Tothis end polyadenylated RNA is prepared from a cell responsive to ICELAP-6, a cDNA library is created from this RNA, the library is dividedinto pools and the pools are transfected individually into cells thatare not responsive to ICE LAP-6. The transfected cells then are exposedto labeled ICE LAP-6. (ICE LAP-6 can be labeled by a variety ofwell-known techniques including standard methods of radio-iodination orinclusion of a recognition site for a site-specific protein kinase.)Following exposure, the cells are fixed and binding of ICE LAP-6 isdetermined. These procedures conveniently are carried out on glassslides.

Pools are identified of cDNA that produced ICE LAP-6-binding cells.Sub-pools are prepared from these positives, transfected into host cellsand screened as described above. Using an iterative sub-pooling andre-screening process, one or more single clones that encode the putativebinding molecule, such as a receptor molecule, can be isolated.

Alternatively a labeled ligand can be photoaffinity linked to a cellextract, such as a membrane or a membrane extract, prepared from cellsthat express a molecule that it binds, such as a receptor molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis("PAGE") and exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative receptor molecule.

Polypeptides of the invention also can be used to assess ICE LAP-6binding capacity of ICE LAP-6 binding molecules, such as receptormolecules, in cells or in cell-free preparations.

Agonists and Antagonists--Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of ICE LAP-6 on cells, such asits interaction with ICE LAP-6-binding molecules such as receptormolecules. An agonist is a compound which increases the naturalbiological functions of ICE LAP-6 or which functions in a manner similarto ICE LAP-6, while antagonists decrease or eliminate such functions.

For example, a cellular compartment, such as a membrane or a preparationthereof, such as a membrane-preparation, may be prepared from a cellthat expresses a molecule that binds ICE LAP-6, such as a molecule of asignaling or regulatory pathway modulated by ICE LAP-6. The preparationis incubated with labeled ICE LAP-6 in the absence or the presence of acandidate molecule which may be an ICE LAP-6 agonist or antagonist. Theability of the candidate molecule to bind the binding molecule isreflected in decreased binding of the labeled ligand. Molecules whichbind gratuitously, i.e., without inducing the effects of ICE LAP-6 onbinding the ICE LAP-6binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to ICE LAP-6 are agonists.

ICE LAP-6-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of ICELAP-6 or molecules that elicit the same effects as ICE LAP-6. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for ICE LAP-6 antagonists is a competitiveassay that combines ICE LAP-6 and a potential antagonist withmembrane-bound ICE LAP-6 receptor molecules or recombinant ICE LAP-6receptor molecules under appropriate conditions for a competitiveinhibition assay. ICE LAP-6 can be labeled, such as by radioactivity,such that the number of ICE LAP-6 molecules bound to a receptor moleculecan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducing ICELAP-6-induced activities, thereby preventing the action of ICE LAP-6 byexcluding ICE LAP-6 from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as receptor molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules. Antoher antagonist is an oligopeptide comprisingthe cleavage site recognition motif for ICE LAP-6.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in--Okano, J. Neurochem. 56: 560 (1991);OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5' coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of ICE LAP-6. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into ICE LAP-6 polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of ICE LAP-6.

Agonists targeted to defective cellular proliferation, including, forexample, cancer cells and solid tumor cells may be used for thetreatment of these diseases. Such targeting may be achieved via genetherapy of using antibody fusions.

Agonists may also be used to treat follicular lymphomas, carcinomasassociated with p53 mutations, autoimmune disorders, such as, forexample, SLE, immune-mediated glomerulonephritis; and hormone-dependenttumors, such as, for example, breast cancer, prostate cancer and ovarycancer; and viral infections, such as, for example, herpesviruses,poxviruses and adenoviruses.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The antagonists may be employed for instance to inhibit the action ofICE LAP-6 polypeptides, for example, in the treatment of Alzheimer'sdisease, Parkinson's disease, rheumatoid arthritis, septic shock,sepsis, stroke, chronic inflammation, acute inflammation, CNSinflammation, osteoporosis, ischemia reperfusion injury, cell deathassociated with cardiovascular disease, polycystic kidney disease,apoptosis of endothelial cells in cardiovascular disease, degenerativeliver disease, MS, ALS, cererbellar degeneration, ischemic injury,myocardial infarction, AIDS, myelodysplastic syndromes, aplastic anemia,male pattern baldness, and head injury damage.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

Gene Therapy

The ICE LAP-6 polynucleotides, polypeptides, agonists and antagoniststhat are polypeptides may be employed in accordance with the presentinvention by expression of such polypeptides in vivo, in treatmentmodalities often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo,and the engineered cells then can be provided to a patient to be treatedwith the polypeptide. For example, cells may be engineered ex vivo bythe use of a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention. Such methods are well-known in theart and their use in the present invention will be apparent from theteachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct then may be isolated and introduced intoa packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the CMVpromoter described in Miller et al., Biotechniques 7:980-990 (1989), orany other promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, RNA polymeraseIII, and β-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the CMV promoter; the respiratorysyncytial virus ("RSV") promoter; inducible promoters, such as the MMTpromoter, the metallothionein promoter; heat shock promoters; thealbumin promoter; the ApoAl promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thyrmidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,YAM, PA 12, T19 14X, VT ·17H2, YCRE, YCRIP, GP⁺ E-86, GP⁺ envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14(1990). The vector may be transduced into the packaging cells throughany means known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplification's, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., cited above.

All parts or amounts set out in the following examples are by weight,unless otherwise specified. As used herein, "CTLs" means cytotoxiclymphocytes.

Unless otherwise stated, size separation of fragments in the examplesbelow was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis ("PAGE") in Sambrook and numerousother references such as, for instance, by Goeddel et al., Nucleic AcidsRes. 8: 4057 (1980).

Unless described otherwise, ligations were accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase ("ligase") per 0.5 μg of DNA.

Example 1

Cloning, Expression and Purification of Human ICE LAP-6 Summary

Members of the ICE/ced-3 gene family are belived to be effectorcomponents of the cell death machinery. Herein this Example, a novelmember of this family designated ICE LAP-6 is characterized. Byphylogenetic analysis, ICE LAP-6 is classified into the Ced-3 subfamilywhich includes Ced-3, Yama/CPP32/apopain, Mch2 and ICE LAP-3/Mch3/CMH-1. ICE LAP-6 contains an active site QACGG (SEQ ID NO:11)pentapeptide, rather than the QACRG (SEQ ID NO:10) pentapeptide sharedby other family members. Overexpression of ICE LAP-6 induces apoptosisin MCF7 breast carcinoma cells. ICE LAP-6 is also proteolyticallyprocessed into an active cysteine protease by granzyme B, an importantcomponent of cytotoxic T cell-mediated apoptosis. Once activated, ICELAP-6 is able to cleave the death substrate poly (ADP-ribose) polymerase(PARP) into signature apoptotic fragments.

Overexpression of ICE LAP-6 in MCF7 breast carcinoma cells induces celldeath and mutation of the putative catalytic cysteine residue abolishesits apoptotic potential. Furthermore, granzyme B directly activates ICELAP-6 and Yama in vitro, suggesting that granzyme B may mediate itscytotoxic effect via activation of several ICE/Ced-3 family members.Once activated, Yama and ICE LAP-6 are both able to cleave the DNArepair enzyme poly (ADP-ribose) polymerase (PARP) into signatureapoptotic fragments. Taken together, these results indicate that ICELAP-6, like other members of the Ced-3 subfamily, likely plays animportant role in the apoptotic mechanism.

Cloning of ICE LAP-6

A cDNA corresponding to the partial open reading frame of ICE LAP-6 wasidentified as a sequence homologous to ICE LAP-3 (Duan, H., et al.(1996) J. Biol. Chem. 271, 35013-35035) on searching database comprisingESTs made by established EST methods (Adams, M. D., et al. (1991)Science 252, 1651-1656; Adams, M. D., et al (1992) Nature 355, 632-634).A novel cDNA clone, encoding a partial open reading frame, wasidentified and showed sequence homology with members of the ICE/ced-3gene family. Of 22 positive clones, 6 clones yielded a 2.3 kb cDNAcontaining an 1252-base pair open reading frame that encoded a novelprotein with a predicted molecular weight of 45.8 kD, designated ICELAP-6 (see FIG. 1). The putative initiator methionine (GCCATGG; Metcodon underlined) was in agreement with the consensus Kozak's sequencefor translation initiation (Kozak, M. (1989) J Cell Biol 108, 229-241).This clone contains an open reading frame encoding the C-terminal 300amino acids of ICE LAP-6. Full length cDNAs were obtained by screeningan oligo-d(T) primed cDNA library of the human chronic myelogenousleukemia cell line K562. Approximately, 1×10⁶ transformants werescreened with a ³² P-labeled DNA fragment generated by PCR,corresponding to nucleotides 615 to 940 of the ICE LAP-6 open readingframe (Sambrook, J., et al (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold spring Harbor Laboratory Press, New York).Double-stranded DNA sequencing was carried out by the dideoxy chaintermination method using modified T7 DNA polymerase (Sequenase, UnitedStates Biochemical Corporation). Sequence alignments were performedusing DNASTAR Megalign software.

Northern Blot

Analyses of Adult and fetal human multiple tissue Northern blots(Clontech) containing 2 μg/lane poly(A)⁺ RNA were hybridized, accordingto the manufacturer's instructions, using the same ³² P-labeled ICELAP-6 probe used for library screening.

Expression Vectors

The DNA inserts encoding the C-terminal FLAG-tagged (ICE LAP-6 flag) orHis6-tagged (ICE LAP-6 His) ICE LAP-6 were generated by PCR andsubcloned into the mammalian expression vector pcDNA3 (Invitrogen). The5' PCR primer (GAACGGGGTACCGCCATGGACGAAGCGGATCGGC) [SEQUENCE ID NO.5]contained a Kpn1 restriction site and the two 3' primers(TGCTCTAGATTACTTGTCATCGTCGTCCTTGTAGTCTGATGTTTTAAAGT TAAGTTTTTTCCGGAG)[SEQUENCE ID NO. 9] or(TGCTCTAGATTAGTGGTGGTGGTGGTGGTGTGATGTTTTAAAGAAAAGT TTTTTCCGGAG)[SEQUENCE ID NO. 6] encoded a FLAG epitope tag (DYKDDDDK) or a His6 tag,respectively. Alteration of the active site cysteine 286 to an alaninewas accomplished by site-directed mutagenesis employing a four-primerPCR-based method (Higuchi, R., et al (1988) Nucleic Acids Research 16,7351-7367). The mutagenetic oligonucleotides wereAAGCTCTTTTTCATCCAGGCCGCGGGTGGGGAGCA GAAGAC [SEQUENCE ID NO. 7] andGTCTTTCTGCTCCCCACCCGCGGCCTGGATGAAAAAAGC [SEQUENCE ID NO. 8]. Thepresence of the introduced mutation and fidelity of PCR replication wereconfirmed by sequence analysis.

Apoptosis Assays

MCF7 breast carcinoma cells were transiently transfected as describedpreviously (Chinnaiyan, A. M., et al (1995) Cell 81, 505-512). Briefly,2.5×10≡MCF7 cells were transfected with 0.25 μg of the reporter plasmidpCMV β-galactosidase plus 1 μg of test plasmid in 6-well tissue culturedishes using lipofectamine as per manufacturer's instructions. Thetransfection was carried out in 1 ml of Opti-MEM Minimal Media(GIBCO-BRL) and after 5 hours, 1 ml of serum-containing growth media wasadded. Two days later, the cells were fixed with 0.5% glutaraldehyde andstained with X-gal for 4 hours. Cells were visualized by phase-contrastmicroscopy. At least 300 β-galactosidase-positive cells were counted foreach transfection (n=3) and identified as apoptotic or nonapoptoticbased on morphological alterations typical of adherent cells undergoingapoptosis including becoming rounded, condensed, and detaching from thedish (Cohen, J. J. (1993) Immunology Today 14, 126-130). Expression andPurification of His6-Tagged Yama and His6-tagged ICE LAP-6 and ³⁵S-labeled Yama and ICE LAP-6 proteins were generated by in vitrotranscription/translation using the TNT kit (Promega) according to theinstructions of the manufacturer; the template plasmids were ICE LAP-6His and Yama His (Tewari, M., et al. (1995) Cell 81, 801-809). Thetranslated proteins were purified by chromatography as describedpreviously (Tewari, M., et al. (1995) Cell 81, 801-809). Activation ofICE LAP-6 and Yama by Granzyme B-Purified in vitro-translated pro-ICELAP-6 or pro-Yama was activated by incubation with granzyme B asdescribed previously (Quan, L. T., et al (1996) PNAS 93, In Press).Briefly, 48 ml of ³⁵ S-labeled protein was incubated with 20 pmole ofpurified granzyme B (22) in a total volume of 50 μl. After 4 hours, 20ml of reaction was removed for SDS-PAGE analysis. 520 pmole of anti-GraB(Quan, L. T., et al (1996) PNAS 93, In Press) was added to the rest ofthe reaction mix to neutralize granzyme B activity. Following a 15 minincubation, 1 ml (150 mg) of purified PARP Tewari, M., et al. (1995)Cell 81, 801-809) was added and the reaction was allowed to proceed for2 hours. The control reaction containing PARP alone or PARP plusgranzyme B and anti-GraB was carried out under identical conditions,except that Yama or ICE LAP-6 was not added. The reaction buffercontained 50 mM Hepes (pH 7.4), 0.1 M NaCi, 0.1% CHAPs, and 10% sucrose.All incubations were carried out at 37° C. in 10 mM DTT. Samples wereanalyzed by immunoblotting with anti-PARP monoclonal antibody C-2-10 asdescribed previously Tewari, M., et al. (1995) Cell 81, 801-809).

ICE LAP-6 is a Novel Member of the ICE/ced-3 Gene Family

A blast search of GenBank protein data base revealed that the predictedprotein sequence of ICE LAP-6 has significant similarity to the membersof the ICE/Ced-3 family, particularly in the regions corresponding tothe active subunits of ICE (Thomberry, N. A.,et al (1992) Nature 356,768-774). In this region, ICE LAP-6 shares 31% sequence identity (55%sequence similarity) with the C. elegans CED-3 protein, 33% identity(52% sequence similarity) with ICE-LAP3, 30% identity (56% similarity)with Mch2a and 29% sequence identity (52% similarity) with Yama. ICELAP-6 also has 25% -28% sequence identity with ICE and the ICE-relatedgenes, ICE rel II and ICE rel III. Phylogenetic analysis of theICE/ced-3 gene family showed that ICE LAP-6 is a member of the Ced-3subfamily which includes Yama, ICE-LAP3, and Mch2 (FIG. 5). Like Ced-3,ICE LAP-6 contains a long N-terminal putative prodomain. Based on thex-ray crystal structure of ICE (Walker, N. P. C. et al, (1994) Cell 78,343-352; Wilson, K. P., et al (1994) Nature 370, 270-275), the aminoacid residues His237, Gly238, Cys285 of ICE are involved in catalysis,while the residues Argl79, Gln283 and Arg341 form a binding pocket forthe carboxylate side chain of the P1 aspartic acid. These six residuesare conserved in all ICE/Ced-3 family members thus far cloned as well asin ICE LAP-6. However, residues that form the P2-P4 binding pockets arenot widely conserved among family members, suggesting that they maydetermine substrate specificity. Surprisingly, ICE LAP-6 contains aunique active site pentapeptide QACGG (SEQ ID No:11), instead of theQACRG (SEQ ID NO:10) shared by other family members.

Distribution of ICE LAP-6

Northern blot analysis revealed that ICE LAP-6 is constitutivelyexpressed in a variety of human tissues. Two ICE LAP-6 mRNA transcriptswere detected. The 2.3 kilobase transcript corresponds to the size ofthe cDNA clones isolated from the K562 library. The other transcript,which is approximately 3 kb, is believed to represent an alternativelyspliced ICE LAP-6 isoform.

Overexpression of ICE LAP-6 in MCF7 Cells Induces Apoptosis

To study the functional role of ICE LAP-6, MCF7 breast carcinoma cellswere transiently transfected with an expression vector encoding thefull-length ICE LAP-6 protein (ICE LAP-6-flag) and subsequently assessedfor apoptotic features. Like the other ICE/Ced-3 family members,expression of ICE LAP-6 caused cell death (FIG. 6). The ICELAP-6-transfected MCF7 cells displayed morphological alterations typicalof adherent cells undergoing apoptosis, becoming rounded, condensed, anddetaching from the dish. ICE LAP-6 induced apoptosis was inhibited bythe broad spectrum ICE inhibitor z-VAD fmk (Pronk, G. J., (1996) Science271, 808-810). To determine whether the amino acid residue Cys286,corresponding to the catalytic Cys285 of ICE, was essential forapoptotic activity, a mutant form of ICE LAP-6 was generated in whichthe cysteine residue was altered to an alanine. MCF7 breast carcinomacells were transiently transfected with the reporter geneb-galactosidase and either C-terminal flag-tagged ICE LAP-6, the mutantversion with the catalytic cysteine residue inactivated (ICE LAP-6 mt)or ICE as described elsewhere herein. Percent apoptotic cells representsthe mean value from three independent experiments. As predicted,overexpression of the mutant form of ICE LAP-6 did not induce apoptoticchanges in MCF7 cells (FIG. 7). Furthermore, these results demonstratethat an ICE/Ced-3 family member containing an active site QACGG (SEQ IDNO:11) pentapeptide (rather than QACRG (SEQ ID NO:10)) may still possessapoptosis-inducing potential and presumably enzymatic activity.

Proteolytic Activation of ICE LAP-6 by Granzyme B

Members of the ICE/ced-3 gene family are synthesized as proenzymes andactivated by proteolytic cleavage at specific aspartate residues to formheterodimeric enzymes. In ICE, this cleavage removes the prodomain andproduces a heterodimeric complex consisting of p20 and p10subunits(Thornberry, N. A.,et al (1992) Nature 356, 768-774). Similarly,activated Yama is comprised of two subunits, pl7 and pl2, which arederived from a 32 kDa proenzyme Nicholson, D. Wet al. (1995) Nature 376,37-43). The mechanism by which death signals activate ICE/Ced-3 familymembers is poorly understood. Recent studies on granzyme B, however,suggest that cytotoxic T cells may utilize this secreted serine proteaseto directly activate members of the ICE/Ced-3 family. It has beendemonstrated that granzyme B can proteolytically activate pro-Yama,generating an active enzyme capable of cleaving the death substrate PARPinto characteristic fragments (Darmon, A. J., et al (1995) Nature 377,446-448). By contrast, ICE, although cleaved by granzyme B, fails to beactivated. Thus, it was determined whether ICE LAP-6 can serve as asubstrate for granzyme B. His6 tagged ICE LAP-6 and Yama were generatedby in vitro transcription/translation, and subsequently purified byNi-affinity chromatography as described elsewhere herein. The purifiedin vitro-translated pro-ICE LAP-6 or pro-Yama was incubated withpurified granzyme B (Hanna, W. L., et al (1993) Protein Expr Purif4,398-404; Quan, L. T., et al (1995) Journal of Biological Chemistry270, 10377-10379). After 4 hours at 37° C., ICE LAP-6 wasproteolytically processed into 3 fragments. The two low molecular weightbands represent the active subunits of ICE LAP-6 and correspond to thep17 and p12 subunits of active Yama. The 32 kDa band is an likelyintermediate, in which only the pro-domain is removed (a similarintermediate is generated in the activation of ICE LAP-3) Duan, H., etal. (1996) J. Biol. Chem. 271, 35013-35035; Chinnaiyan, A. M., et al.1996) Journal of Biological Chemistry 271, 4573-4576). Next, granzymeB-mediated cleavage of ICE LAP-6 was assessed for generation of anactive enzyme by assaying for PARP cleavage. PARP is proteolyzed duringmany forms of apoptosis, and the enzyme(s) responsible is likely of theICE/Ced-3 family. To exclude the possibility of direct cleavage of PARPby granzyme B, granzyme B-processed ICE LAP-6 and Yama were incubatedwith a selective inhibitor of granzyme B (anti-GraB). Both granzymeB-processed Yama and ICE LAP-6 were active as determined by theirability to cleave PARP. Unlike ICE, ICE LAP-6 and other members of theCed-3 subfamily are able to cleave the PARP into signature apoptoticfragments (Tewari, M., et al. (1995) Cell 81, 801-809;Fernandes-Alnemri, T., et al. (1994) J. Biol. Chem. 269, 30761-30764-Nicholson, D. Wet al. (1995) Nature 376, 37-43; Fernandes-Alnemri, T.,et al. (1995) Cancer Research 55, 6045-6052; Lippke, J. A., et al.(1996) The Journal of Biological Chemistry 271, 1825-1828;Fernandes-Alnemri, T., et al. (1995) Cancer Res 55, 2737-2742).

Provided by the present invention is a novel member of the ICE/Ced-3family of cysteine proteases. ICE LAP-6 has a unique active site QACGG(SEQ ID NO:11) pentapeptide and is classified in the subfamily mostrelated to Ced-3 and Yama. Ectopic expression of ICE LAP-6 in mammaliancells causes apoptosis.

Importantly, ICE LAP-6, like Yama, was directly activated by granzyme Bin vitro, suggesting that cytotoxic T cells may mediate apoptosis byactivating more than one ICE/Ced-3 family member in susceptible targetcells. Yama, ICE-LAP3, and now ICE LAP-6, have been shown to beproteolytically activated by apoptotic stimuli.

Example 2

Gene Therapeutic Expression of Human ICE LAP-6

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted--the chunks of tissue remain fixed to the bottom of theflask--and fresh media is added (e.g., Ham's F12 media, with 10% FBS,penicillin and streptomycin). The tissue is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerges. The monolayer istrypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes forcloning a fragment to be expressed. The digested vector is treated withcalf intestinal phosphatase to prevent self-ligation. Thedephosphorylated, linear vector is fractionated on an agarose gel andpurified.

ICE LAP-6 cDNA capable of expressing active ICE LAP-6, is isolated. Theends of the fragment are modified, if necessary, for cloning into thevector. For instance, 5' overhanging may be treated with DNA polymeraseto create blunt ends. 3' overhanging ends may be removed using S1nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backboneand the ICE LAP-6 fragment are mixed together and joined using T4 DNAligase. The ligation mixture is used to transform E. coli and thebacteria are then plated onto agar-containing kanamycin. Kanamycinphenotype and restriction analysis confirm that the vector has theproperly inserted gene.

Packaging cells are grown in tissue culture to confluent density inDulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS),penicillin and streptomycin. The vector containing the ICE LAP-6 gene isintroduced into the packaging cells by standard techniques. Infectiousviral particles containing the ICE LAP-6 gene are collected from thepackaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriateincubation period media is harvested from the plates of confluentproducer cells. The media, containing the infectious viral particles, isfiltered through a Millipore filter to remove detached producer cells.The filtered media then is used to infect fibroblast cells. Media isremoved from a sub-confluent plate of fibroblasts and quickly replacedwith the filtered media. Polybrene (Aldrich) may be included in themedia to facilitate transduction. After appropriate incubation, themedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his, toselect out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone orafter having been grown to confluence on microcarrier beads, such ascytodex 3 beads. The injected fibroblasts produce ICE LAP-6 product, andthe biological actions of the protein are conveyed to the host.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 11                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 416 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #Arg Cys Arg Leu Arg Leurg Arg Leu Leu Arg                                    #                 15                                                          #Asp Val Leu Leu Ser Argal Asp Gln Leu Trp                                    #             30                                                              #Ile Gln Arg Ala Gly Seris Met Ile Glu Asp                                    #         45                                                                  #Ile Ile Asp Leu Glu Thrln Ala Arg Gln Leu                                    #     60                                                                      #Ser Cys Leu Glu Asp Threu Pro Leu Phe Ile                                    # 80                                                                          #Thr Asn Arg Gln Ala Glyla Ser Phe Leu Arg                                    #                 95                                                          #Thr Pro Val Val Leu Arghr Leu Glu Asn Leu                                    #            110                                                              #Pro Glu Thr Pro Arg Proro Glu Val Leu Arg                                    #        125                                                                  #Val Gly Ala Leu Glu Serly Gly Phe Gly Asp                                    #    140                                                                      #Leu Ser Met Glu Pro Cyssp Leu Ala Tyr Ile                                    #160                                                                          #Phe Cys Arg Glu Ser Glyle Asn Asn Val Asn                                    #                175                                                          #Cys Glu Lys Leu Arg Argly Ser Asn Ile Asp                                    #            190                                                              #Val Lys Gly Asp Leu Thris Phe Met Val Glu                                    #        205                                                                  #Leu Ala Arg Gln Asp Hiseu Ala Leu Leu Glu                                    #    220                                                                      #Leu Ser His Gly Cys Glnys Val Val Val Ile                                    #240                                                                          #Tyr Gly Thr Asp Gly Cyshe Pro Gly Ala Val                                    #                255                                                          #Phe Asn Gly Thr Ser Cysys Ile Val Asn Ile                                    #            270                                                              #Phe Ile Gln Ala Cys Glyys Pro Lys Leu Phe                                    #        285                                                                  #Ala Ser Thr Ser Pro Gluis Gly Phe Glu Val                                    #    300                                                                      #Asp Ala Thr Pro Phe Glner Asn Pro Glu Pro                                    #320                                                                          #Ala Ile Ser Ser Leu Prohe Asp Gln Leu Asp                                    #                335                                                          #Thr Phe Pro Gly Phe Valhe Val Ser Tyr Ser                                    #            350                                                              #Tyr Val Glu Thr Leu Aspys Ser Gly Ser Trp                                    #        365                                                                  #Asp Leu Gln Ser Leu Leurp Ala His Ser Glu                                    #    380                                                                      #Gly Ile Tyr Lys Gln Metla Val Ser Val Lys                                    #400                                                                          #Leu Phe Phe Lys Thr Serhe Leu Arg Lys Lys                                    #                415                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1578 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - GCCATGGACG AAGCGGATCG GCGGCTCCTG CGGCGGTGCC GGCTGCGGCT GG - #TGGAAGAG         60                                                                          - CTGCAGGTGG ACCAGCTCTG GGACGTCCTG CTGAGCCGCG AGCTGTTCAG GC - #CCCATATG        120                                                                          - ATCGAGGACA TCCAGCGGGC AGGCTCTGGA TCTCGGCGGG ATCAGGCCAG GC - #AGCTGATC        180                                                                          - ATAGATCTGG AGACTCGAGG GAGTCAGGCT CTTCCTTTGT TCATCTCCTG CT - #TAGAGGAC        240                                                                          - ACAGGCCAGG ACATGCTGGC TTCGTTTCTG CGAACTAACA GGCAAGCAGG AA - #AGTTGTCG        300                                                                          - AAGCCAACCC TAGAAAACCT TACCCCAGTG GTGCTCAGAC CAGAGATTCG CA - #AACCAGAG        360                                                                          - GTTCTCAGAC CGGAAACACC CAGACCAGTG GACATTGGTT CTGGAGGATT CG - #GTGATGTC        420                                                                          - GGTGCTCTTG AGAGTTTGAG GGGAAATGCA GATTTGGCTT ACATCCTGAG CA - #TGGAGCCC        480                                                                          - TGTGGCCACT GCCTCATTAT CAACAATGTG AACTTCTGCC GTGAGTCCGG GC - #TCCGCACC        540                                                                          - CGCACTGGCT CCAACATCGA CTGTGAGAAG TTGCGGCGTC GCTTCTCCTC GC - #TGCATTTC        600                                                                          - ATGGTGGAGG TGAAGGGCGA CCTGACTGCC AAGAAAATGG TGCTGGCTTT GC - #TGGAGCTG        660                                                                          - GCGCGGCAGG ACCACGGTGC TCTGGACTGC TGCGTGGTGG TCATTCTCTC TC - #ACGGCTGT        720                                                                          - CAGGCCAGCC ACCTGCAGTT CCCAGGGGCT GTCTACGGCA CAGATGGATG CC - #CTGTGTCG        780                                                                          - GTCGAGAAGA TTGTGAACAT CTTCAATGGG ACCAGCTGCC CCAGCCTGGG AG - #GGAAGCCC        840                                                                          - AAGCTCTTTT TCATCCAGGC CTGTGGTGGG GAGCAGAAAG ACCATGGGTT TG - #AGGTGGCC        900                                                                          - TCCACTTCCC CTGAAGACGA GTCCCCTGGC AGTAACCCCG AGCCAGATGC CA - #CCCCGTTC        960                                                                          - CAGGAAGGTT TGAGGACCTT CGACCAGCTG GACGCCATAT CTAGTTTGCC CA - #CACCCAGT       1020                                                                          - GACATCTTTG TGTCCTACTC TACTTTCCCA GGTTTTGTTT CCTGGAGGGA CC - #CCAAGAGT       1080                                                                          - GGCTCCTGGT ACGTTGAGAC CCTGGACGAC ATCTTTGAGC AGTGGGCTCA CT - #CTGAAGAC       1140                                                                          - CTGCAGTCCC TCCTGCTTAG GGTCGCTAAT GCTGTTTCGG TGAAAGGGAT TT - #ATAAACAG       1200                                                                          - ATGCCTGGTT GCTTTAATTT CCTCCGGAAA AAACTTTTCT TTAAAACATC AT - #AAGGCCAG       1260                                                                          - GGCCCCTCAC CCTGCCTTAT CTTGCACCCC AAAGCTTTCC TGCCCCAGGC CT - #GAAAGAGG       1320                                                                          - CTGAGGCCTG GACTTTCCTG CAACTCAAGG ACTTTGNAGC CGGCACAGGG TC - #TGCTCTTT       1380                                                                          - CTCTGCCAGT GACAGACAGG CTCTTAGCAG CTTCCAGATT GACGACAAGT GC - #TGAACAGT       1440                                                                          - GGAGGAAGAG GGACAGATGA ATGCCGTGGA TTGCACGTGG NCTCTTGAGC AG - #TGGCTGGT       1500                                                                          - CCAGGGCTAG TGACTTGGTG TCCCATGATC CCTGTGTTGG TCTCTAGGAG CA - #GGGATTAA       1560                                                                          #1578              AT                                                         - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 639 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - CTGACTGCCA AGAAAATGGT GCTGGCTTTG CTGGAGCTGG CGCGGCAGGA CC - #ACGGTGCT         60                                                                          - CTGGACTGCT GCGTGGTGGT CATTCTCTCT CACGGCTGTC AGGCCAGCCA CC - #TGCAGTTC        120                                                                          - CCAGGGGCTG TCTACGGCAC AGATGGATGC CCTGTGTCGG TCGAAAAGAT TG - #TGAACATC        180                                                                          - TTCAATGGGA CCAGCTGCCC CAGCCTGGGA GGGAAGCCCA AGCTCTTTTT CA - #TCCAGGCC        240                                                                          - TGTGGTGGGG AGCAGAAAGA CCATGGGTTT GAGGTGGCCT CCACTTCCCC TG - #AAGACGAG        300                                                                          - TCCCCTGGCA GTAACCCCGA GCCAGATGCC ACCCCGTTCC AGGAAGGTTT GA - #GGACCTTC        360                                                                          - GACCAGCTGG ACGCCATATC TAGTTTGCCC ACACCCAGTG ACATCTTTGT GT - #CCTACTCT        420                                                                          - ACTTTCCCAG GTTTTGTTTC CTGGAGGGAC CCCAAGAGTG GCTCCTGGTA CG - #TTGAGACC        480                                                                          - CTGGACGACA TCTTTGAGCA GTGGGCTCAC TCTGAAGACC TGCAGTCCCT CC - #TGCTTAGG        540                                                                          - GTCGCTAATG CTGTTTCGGT GAAAGGGATT TATAAACAGA TGCCTGGTTG CT - #TTAATTTC        600                                                                          #   639            TCTT TTAAAACATC ATAAGGCAG                                  - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 203 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #Gln Asp His Gly Ala Leueu Glu Leu Ala Arg                                    #                 15                                                          #Gly Cys Gln Ala Ser Hisal Ile Leu Ser His                                    #             30                                                              #Asp Gly Cys Pro Val Serla Val Tyr Gly Thr                                    #         45                                                                  #Thr Ser Cys Pro Ser Leusn Ile Phe Asn Gly                                    #     60                                                                      #Ala Cys Gly Gly Glu Glneu Phe Phe Ile Gln                                    # 80                                                                          #Ser Pro Glu Asp Glu Serlu Val Ala Ser Thr                                    #                 95                                                          #Pro Phe Gln Glu Gly Leulu Pro Asp Ala Thr                                    #            110                                                              #Ser Leu Pro Thr Pro Sereu Asp Ala Ile Ser                                    #        125                                                                  #Gly Phe Val Ser Trp Argyr Ser Thr Phe Pro                                    #    140                                                                      #Thr Leu Asp Asp Ile Pheer Trp Tyr Val Glu                                    #160                                                                          #Ser Leu Leu Leu Arg Valer Glu Asp Leu Gln                                    #                175                                                          #Lys Gln Met Pro Gly Cysal Lys Gly Ile Tyr                                    #            190                                                              #Methe Asn Phe Leu Arg Lys Lys Leu Phe Phe                                    #        200                                                                  - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #        34        TGGA CGAAGCGGAT CGGC                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - TGCTCTAGAT TAGTGGTGGT GGTGGTGGTG TGATGTTTTA AAGAAAAGTT TT - #TTCCGGAG         60                                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   41             AGGC CGCGGGTGGG GAGCAGAAGA C                               - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #    39            CCCG CGGCCTGGAT GAAAAAAGC                                  - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 66 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - TGCTCTAGAT TACTTGTCAT CGTCGTCCTT GTAGTCTGAT GTTTTAAAGT TA - #AGTTTTTT         60                                                                          #           66                                                                - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                -  Gln Ala Cys Arg Gly                                                          1               5                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                -  Gln Ala Cys Gly Gly                                                          1               5                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide encoding a polypeptide having at least 30 consecutiveamino acids contained in SEQ ID NO:1.
 2. An isolated polynucleotidecomprising a polynucleotide encoding a polypeptide having at least 50consecutive amino acids contained in SEQ ID NO:1.
 3. An isolatedpolynucleotide comprising a polynucleotide encoding the same maturepolypeptide encoded by the cDNA contained in 1095150deposited with theAmerican Type Culture Collection.
 4. An isolated polynucleotide whichencodes a polypeptide comprising the amino acid sequence set forth inSEQ ID NO:
 1. 5. The isolated polynucleotide of claim 4 comprising theregion of the polynucleotide set forth in SEQ ID NO:2 encoding thepolypeptide of SEQ ID NO:
 1. 6. The isolated polynucleotide of claim 4comprising an RNA transcript of the entire length of SEQ ID NO:2.
 7. Theisolated polynucleotide of claim 4 comprising an RNA transcript of theentire coding region of SEQ ID NO:2.
 8. An expression vector comprisingthe polynucleotide of claim
 4. 9. An isolated host cell comprising thevector of claim
 8. 10. A process for producing a polypeptide comprising:culturing the host cell of claim 9 such that the polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO:1 is produced.
 11. Aprocess for producing a cell which expresses a polypeptide comprisingtransforming or transfecting the cell with the vector of claim 8 suchthat the cell expresses the polypeptide comprising the amino acidsequence set forth in SEQ ID NO:1.
 12. The isolated polynucleotide ofany one of claims 1-4 wherein the polynucleotide is DNA.
 13. Theisolated polynucleotide of any one of claims 4 wherein thepolynucleotide is RNA.
 14. An isolated polynucleotide comprising theentire nucleotide sequence set forth in SEQ ID NO:2.
 15. An isolatedpolynucleotide which is complementary to the entire length of any one ofthe isolated polynucleotides of claims 3-7 and 14.